Abscisic acid improves drought resilience, growth, physio-biochemical and quality attributes in wheat (Triticum aestivum L.) at critical growth stages

Wheat is an important staple crop not only in Pakistan but all over the globe. Although the area dedicated to wheat cultivation expands annually, the quantity of wheat harvested is declining due to various biotic and abiotic factors. Global wheat production and output have suffered as a result of the drought, which is largely driven by a lack of water and environmental factors. Organic fertilizers have been shown to reduce the severity of drought. The current research was conducted in semi-arid climates to mitigate the negative effects of drought on wheat during its critical tillering (DTS), flowering (DFS), and grain filling (DGFS) stages through the application of three different abscisic acid treatments: ABA0 (0 mgL−1) control, ABA1 (100 mgL−1) and ABA2 (200 mgL−1). Wheat growth and yield characteristics were severely harmed by drought stress across all critical development stages, with the DGFS stage being particularly vulnerable and leading to a considerable loss in yield. Plant height was increased by 24.25%, the number of fertile tillers by 25.66%, spike length by 17.24%, the number of spikelets per spike by 16.68%, grain count per spike by 11.98%, thousand-grain weight by 14.34%, grain yield by 26.93% and biological yield by 14.55% when abscisic acid (ABA) was applied instead of the control treatment. Moreover, ABA2 increased the more physiological indices (water use efficiency (36.12%), stomatal conductance (44.23%), chlorophyll a (24.5%), chlorophyll b (29.8%), transpiration rate (23.03%), photosynthetic rate (24.84%), electrolyte leakage (− 38.76%) hydrogen peroxide (− 18.09%) superoxide dismutase (15.3%), catalase (20.8%), peroxidase (− 18.09%), and malondialdehyde (− 13.7%)) of drought-stressed wheat as compared to other treatments. In the case of N, P, and K contents in grain were maximally improved with the application of ABA2. Through the use of principal component analysis, we were able to correlate our results across scales and provide an explanation for the observed effects of ABA on wheat growth and production under arid conditions. Overall, ABA application at a rate of 200 mgL−1 is an effective technique to boost wheat grain output by mitigating the negative effects of drought stress.

three replications.Furthermore, full irrigation was used as a control (CK) and plants in the control treatment were sprayed with the same amount of deionized water 43 .
In this experiment, used Galaxy 2013 wheat cultivar selected after the screening of 5 different verities such as Punjab 2011, Faisalabad 2008, Lasani 2008, 8200 and Galaxy 2013.Moreover, this verity is also recommended in this semi-arid region by Regional Agricultural Research Institute (RARI) Bahawalpur 44 .On November 10th, 2019, wheat seeds were sown in the 36 pots (26 × 29 cm; three for each application) filled with six kg of soil and seeds were purchased from RARI Bahawalpur.Fertilizers 0.52 g N, 0.35 g P, and 0.23 g K were added in each pot according to the recommended dose of N, P, and K (160-115-75 kg/ha) in wheat.The soils physiochemical composition is shown in Table 1.In the event of inclement weather, a transparent plastic cover was put over the wire enclosure to shield the plants within.Up to the point of complete emergence, all containers were watered uniformly.After 20 days of seeding, a total of four plants were kept in each container by removing the excess.A translucent plastic canopy was placed over the wire-house to protect the crop from rainfall when needed.All the containers were watered equally until the plants fully emerged.

Growth and yield parameters
The growth, yield, and various attributes of wheat were measured, including spike length (cm), number of grains per spike, number of spikelets per spike, number of fertile tillers, 1000-grain weight (g), plant height (cm), organic yield per plant (g), grain yield per plant (g), and harvest index.These measurements were obtained using established procedures and protocols.The yield of wheat plants was obtained by physically harvesting them at the end of their life cycle.Similarly, to measure the height of the plants, three plants were randomly chosen from each pot and their height was measured from the surface of the soil to the spikelet using a ruler calibrated in millimetres and centimetres.To measure the length of the spike and the number of grains per spike, use a centimetre scale to measure the distance from the base to the terminal spikelet of three randomly chosen plants.Additionally, count the number of grains in ten randomly selected clusters of seeds.In addition, the digital balance was used to measure the 1000-grain weight, taking advantage of its precision of 0.01 g.The following formula was used to determine the harvest index (HI):

Determination of physiological parameters
In accordance with the method employed by Khalilzadeh et al. 48, 0.5 g of recently harvested leaf tissue was slowly mixed with 10 mL of acetone (80%) to determine the chlorophyll levels.The mixture was then subjected to centrifugation at a speed of 400 rpm for a duration of 10 min.The spectrophotometer recorded the absorbance at wavelengths of 645 nm, 663 nm, and 470 nm.The chlorophyll contents were acquired using the following method:  47 defined a mathematical equation for determining the water usage efficiency (WUE, g pot −1 mm −1 ) as follows: Automatic porometer MK-3 Delta-T Devices, BC, England, was used to measure the transpiration rate and stomatal conductance (SC).It was measured from 5 leaves per pot between 11:00 and 13:00 h, when atomosphre was cleared and stomata were fully opened and photosynthetic activity were high 49 .

Determination of electrolyte leakage and hydrogen peroxide concentration
The shoot samples were vertically inserted into tubes to measure electrolyte leakage percentage and heated in distilled water at 32 °C for 2 h.After this step, the electrical conductivity (EC) of the solution was determined as EC1.Subsequently, the solution was heated at a constant temperature of 121 °C for 20 min.EL was calculated using a previously defined equation 50 .
To determine the concentration of hydrogen peroxide (H 2 O 2 ), a 50 mg leaf tissue was mixed with 3.0 mL of phosphate buffer solution and centrifuged at 6000g for 30 min at 4 °C.Then an upper layer of the centrifuges sample was mixed with 1.0 mL of (0.1%) titanium sulphate by centrifuging at 6000g for 20 min at 4 °C.Specifically, the absorbance was measured at 410 nm.The extinction coefficient for H 2 O 2 was found to be 0.28 mol −1 cmss −1 .

Estimation of antioxidant enzymes
The activity of superoxide dismutase (SOD) was measured using the procedure outlined by Giannopolitis and Ries 51 . 2 µM riboflavin was added to a 3 mL reaction mixture containing 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 µM nitro blue tetrazolium, 0.1 µM EDTA, and 0-100 µl of enzyme extract 51 .The tubes were shaken and lighted with a 15-Watt fluorescent bulb.The reaction was allowed to proceed for a duration of 10 min.Subsequently, the light source was turned off and the absorbance was measured at a wavelength of 560 nm.A single unit of SOD activity was determined as the quantity of enzyme needed to induce a 50% decrease in the rate of nitroblue tetrazolium chloride reduction.
An approach for determining CAT activity was suggested by Hwang et al. 52 , which involves monitoring the rate of H 2 O 2 breakdown at 240 nm.Modifications were made to the guaiacol oxidation procedure reported by Maechlay and Chance 53 to determine POD activity.The reaction mix included 50 mM potassium phosphate buffer (pH 6.1), 1% guaiacol, 0.4% H 2 O 2 , and extracted enzyme, and it took up a total volume of 3 ml.At 470 nm, an increase in absorbance (E = 25.5 mM −1 cm −1 ) was seen owing to oxidation of guaiacol 54 .MDA contents were estimated as described by Wu et al. 55 .

Grain quality parameters
The levels of nitrogen (N), phosphorus (P), and potassium (K) were examined to evaluate the quality of the grain.The estimation of total nitrogen from wheat seeds was conducted using the Micro Kjeldahl's technique, as described by Piper 56 .To assess the P content in wheat seed, the vanado-phosphate molybdate yellow colour technique proposed by Kitson and Mellon 57 was used on a di-acid extract.To assess the K level in wheat seed, a flame photometer with a di-acid extract method, as reported by Piper 56 , was used.

Statistical analysis
The analysis of variance (ANOVA) was calculated using STATISTIX software (version 8.1) on the present data with the least significant difference (Tukey's HSD) at the 5% probability level to compare the means of the variables 58 .A biplot graph was created in Origin Pro 9.1 to visualize the principal component analysis (PCA) findings (Fig. 1).

Plant height and spike length
The imposition of drought stress caused a significant reduction in plant height by 34.9%, 32.1%, and 24.3%, and spike length by 38.6%, 33.1%, and 26.6% at the DTS, DFS, and DGFS, respectively as compared to CK (no drought at any stage).However, the ABA applications (ABA 1 and ABA 2 ) mitigated the impact of drought stress, leading to an increase in plant height and spike length by 18.7%, 24.2%, and 13.5%, 17.2%, respectively as compared to control treatment ABA 0 (Table 2).

Yield attributes
The drought conditions at specific growth stages (DTS, DFS, and DGFS) lowered the spikelet's numbers per spike (19.8, 36.7, and 51.1%), the number of fertile tillers (24.6, 40.5, and 57.4%), the grain number per spike (18.8, 27.1, and 38.1%), the 1000-grain weight (12.7, 25.4, and 35.5%), the grain yield (13.0, 31.1, and 40.4%), the biological yield (17.1, 39.0, and 76.5%), and the harvest index (3.3,4.9, and 20.6%) as compared to control (CK) when no drought imposed at any stages.Under both drought and control conditions, ABA demonstrated a positive effect, mitigating the adverse effects of drought and enhancing the values of the aforementioned parameters.Notably, a foliar application of ABA 2 at 200 mgL −1 resulted in a significantly higher grain yield (26.9%) compared to the application of ABA 1 at 100 mgL −1 and ABA 0 control treatment (Table 3).Moreover, ABA foliar www.nature.com/scientificreports/treatment effectively reduced the negative impact of drought and simultaneously enhanced the potential of various yield-related characteristics of wheat, including NSPS, NFT, NGPS, 1000Gwt, GY, BY, and HI (Tables 2, 3).

Chlorophyll concentrations
The application of ABA had a significant impact on the chlorophyll content (a and b) of wheat leaves under drought as shown in Fig. 2. Compared to the control (ABA 0 ) with 100 and 200 mgL −1 concentrations of ABA Table 2.The effect of abscisic acid on plant height (PH, cm), spike length (SL, cm), number of spikelets per spike (NSPS), number of grains per spike (NGPS), number of fertile tiller (NFT) of wheat under different drought episodes.ABA 0 , ABA 1 and ABA 2 treatments indicates 0 mgL −1 (control), 100 mgL −1 , and 200 mgL −1 of ABA respectively.Ck, DTS, DFS, and DGFS treatments indicates no drought at any stage, drought at tillering, drought at flowering and grain filling stages respectively.There were no significant differences at the 5% probability level among the means that share the same letter case.NS = non-significant, * = significant at p ≤ 0.05, ** = significant at p ≤ 0.01 and *** = significant at p ≤ 0.001.Significant values are in bold.

Water use efficiency and stomatal conductance
ABA applications (ABA 1 and ABA 2 ) significantly reduced the drought impact by 29.8% and 44.2%, respectively, as compared to the control (ABA 0 ).The application of ABA at different development stages resulted in increased efficiency of wheat water consumption under drought (Fig. 2).Water use efficiency (WUE) decreased by 31.1%,40.8%, and 32.1% under drought at DTS, DFS, and DGFS, respectively, in comparison to the control (CK) when no drought was imposed at any stages.However, the application of ABA (ABA 1 and ABA 2 ) significantly mitigated the impact of drought, reducing the WUE decline by 20.2% and 36.1%,respectively, compared to the non-ABA treated plants.Additionally, stomatal conductance (m mol m −2 s −1 ) was reduced by 13.0%, 24.3%, and 37.4% under drought stress at DTS, DFS, and DGFS, respectively, in comparison to the control treatment (CK).Nevertheless, ABA 1 and ABA 2 applications significantly alleviated the drought impact, reducing the stomatal conductance decline by 29.8% and 44.2%, respectively, compared to the control treatment ABA 0 .

Antioxidant enzymatic activities
Under drought stress, H 2 O 2 and EL percentage were elevated by 17.6%, 22.4%, and 29.8% for H 2 O 2 and 19.5%, 13.8%, and 12.9% for EL, at the DTS, DFS, and DGFS stages of wheat, respectively, as compared to control when no drought was imposed at any stage (Fig. 3C,D).However, the application of ABA 1 and ABA 2 (Fig. 3C,D) Table 3.The effect of ABA on 1000-grain weight (1000 Gwt, g), grain yield per plant (GY, g), biological yield per plant (BY, g), harvesting index (HI, %) of wheat under different drought episodes.ABA 0 , ABA 1 and ABA 2 treatments indicates 0 mgL −1 (control), 100 mgL −1 , and 200 mgL −1 of ABA respectively.Ck, DTS, DFS, and DGFS treatments indicates no drought at any stage, drought at tillering, drought at flowering and grain filling stages respectively.There were no significant differences at the 5% probability level among the means that share the same letter case.NS = non-significant, * = significant at p ≤ 0.05, ** = significant at p ≤ 0.01 and *** = significant at p ≤ 0.001.Significant values are in bold.The application of ABA under drought conditions had a significant impact on the antioxidant enzymatic activities of wheat leaves, as demonstrated in Fig. 4. Drought stress influence the enzymatic activities of SOD, CAT, POD, and MDA as compare to control treatments (CK and ABA 0 ).However, the addition of ABA 1 and ABA 2 during the DTS, DFS, and DGFS stages substantially modulated these activities when compared with ABA 0 .SOD and CAT activities (Fig. 4A,C) were decreased by 38.5%, 34.0%, 28.0%, and 3.1%, 8.1%, 11.4%, respectively, when compared to CK, no drought was imposed.Moreover, ABA 1 and ABA 2 applications significantly increased SOD and CAT activities by 7.3%, 15.3%, and 12.0%, 20.8%, respectively, in comparison to the control (ABA 0 ) under drought conditions.

Principal component analysis
Principal component analysis (PCA) has been widely employed to analyze the variations and associations among different growth, physiological, morphological, biochemical, yield, and quality-linked attributes of wheat under the application of nutrient 44,59 .In this study, we also conducted PCA to evaluate the variability and associations among agronomic traits and the distribution of treatments (see Figs. 6 and 7).In Fig. 6, agronomic traits which are denoted with different vectors were used and spread among the different clades.In Fig. 7, both agronomic traits and treatments were used and a biplot was created.The cumulative variance of the first two PCs was 91.1% of the total variance (Fig. 6, 7).The first two PCs (PC1 = 76%, and PC2 = 15.1%) were significant.All the studied parameters were the major contributing factors in PC1 except EL%, MDA, POD, SOD, H 2 O 2 , HI, and CAT.Similarly, in PC2, SOD was the main factor and showed the highest loading values (Fig. 6).Projection of traits showed that MDA, H 2 O 2 , and POD were strongly related to each other.Similarly, in the PCA-biplot, the treatments scattered away from each other and indicated clear differences among them (Fig. 7).Based on PCA results, these parameters can be utilized in future breeding programs.

Discussion
The main goal of this study was to identify the most effective doses of ABA for foliar application, aiming to expedite wheat growth and alleviate the adverse impacts of drought (Fig. 1).Notably, substantial improvements in growth attributes were observed when applying a dosage of ABA 200 mgL −1 under stressful conditions.Reduced wheat harvests may be attributed to drought stress, a global growth limitation 60 .Low water availability might stunt a plants growth 61 .Wheat plant development is drastically altered by ABA treatments.Hussain et al. 62 found that water stress significantly affected cell division.The disruption of protoplasmic processes caused by diminished turgidity and dehydration leads to less cell division and shorter plants as a result.Plant growth regulators are very useful here since they increase plant height.Drought stress, at any stage of the development cycle, reduced the plants height 63 .Possible hormonal changes during drought stress might have a significant impact on plant height 64 .Our result also in line with the previous researches and showed that the height of wheat was increased by ABA 1 and ABA 2 application as compared to the control under stress.
Plants with longer spikes are more likely to produce a greater number of spikelets and a better production 44 .Lack of water in many plant functions, as described by Fahad et al. 65 reduces the number of spike lengths.According to Iqbal 66 , using ABA improve the nutritional availability, which in turn results in longer spikes.Specifically, proline synthesis is increased by ABA under dry circumstances, which in turn increases spike length and enhances plant metabolic processes 67 .Same pattern observed in our study that ABA treatment increased spike numbers under drought conditions when compared with control.
Reduced water availability in the plant as a result of drought stress reduces the number of fertile tillers.When plants have less access to water, their metabolic activities slow down, resulting in fewer tillers 68 .The number of tillers is reduced by 14.6% during the tillering stage, 20.4% during the blooming stage, and 27.4% during the grain-filling stage when drought is applied.In the face of drought, ABA showed its greatest effectiveness in increasing the number of tillers.According to Sabagh et al. 69 , the capacity of ABA to enhance plant development is context-dependent.
The economic yield is very sensitive to the quantity of spikelets per spike.A decrease in spike length as a consequence of drought stress leads to a drop in spikelet density 61 .Drought during wheat grain-filling phase resulted in a decrease in spikelet production.The outcomes of ABA are equally noteworthy.Wheat plants with ABA 2 application had the most spikelets per spike.He et al. 28 found that plant hormones boost plant vegetative development by inducing the synthesis of chemicals that mitigate the negative effects of drought.According to research by Raza et al. 71 grains per spike were significantly impacted by drought stress at any growth stage.The quantity of grains produced per spike shortens and the length of the spike under drought stress.According to Tripathi et al. 72 , ABA may stimulate more robust plant development.Plant growth regulators boost proline production, which benefits plant health.In comparison to the control, seed priming with ABA 1 and ABA 2 increased grain yield.Zulfiqar et al. 10 found that drought stress reduces grain weight, due to stunted development and reduced nutrient absorption by plants under water deficit.The molecule Indole-3-acetic acid is produced by ABA, and proline synthesis also aids in raising the drought threshold 73 .Plants that produce growth-regulating chemicals have better metabolic activities and a greater sink capacity, both of which contribute to a heavier 1000-grain yield.ABA 1 and ABA 2 in the current study had positively increase 1000-grain weight.
Plants under drought stress saw a drop in biological output at the tillering stage, flowering stage, and the grain-filling stage.Drought-related reductions in grain weight and plant height reduced biological yield 44,62 .ABA promotes optimal plant development 74 .ABA aids in enhancing nutrient absorption, which in turn imparts physiological changes in plant development, which in turn leads to a greater biological yield.According to Tripathi et al. 72 research, the capacity of various ABA applications to stimulate plant development varies.Same trend was seen in this research for biological yield when ABA 1 and ABA 2 were applied.
Grain yield was reduced due to drought stress in every drought stages.At the anthesis stage, Zulfiqar et al. 10 found that drought stress was most damaging to grain output.Water scarcity stress also reduces grain weight and yield by disrupting the absorption of nutrients.Under drought circumstances, the grain yield of ABA 1 and ABA 2 was raised as compared to the control treatment.These results have similar pattern with the previous studies.
As the temperature and humidity were both regulated, water use efficiency improved.When comparing tillering, blooming, and grain-filling drought, the control treatment had greater WUE.According to Lu et al. 75 WUE improved when their interconnected units were used to their fullest extent.The use of ABA in wheat crops has been shown to increase both plant function and plant WUE.ABA 1 and ABA 2 foliar application, respectively, resulted higher WUE compared to the control when tested under drought circumstances which were matched with previous studies.
The chlorophyll level of a leaf is crucial to the plants ability to make nourishment.When leaf area is reduced, chlorophyll concentration is also reduced 76 .In addition, researchers 44,71 found that drought stress reduced the plant's leaf area (LA), and a drop in LA resulted in a decrease in chlorophyll concentration.According to research by Raza et al. 60 production of ROS that are harmful to chloroplasts increases in response to drought stress.ABA administration resulted in enhanced metabolic processes, decreased ROS levels, and increased cell proliferation.When ABA 1 and ABA 2 applied, the chlorophyll content increased, compared to the control in a drought experiment.
Furthermore, oxidative stress can detrimentally affect a plants biological activity through lipid peroxidation and nucleic acid damage 77,78 .It is essential to strike a balance between ROS generation and breakdown for optimal plant development 78 .For this reason, plants are vulnerable to oxidative stress since they cannot detoxify ROS 79,80 .Our findings revealed that during drought stress, plant EL, H 2 O 2 concentration, and POD activity all increased, whereas SOD and CAT activities decreased.In addition, ABA role in maintaining and restoring wheat plant cell membranes contributed considerably to a reduction in ROS.According to Wang et al. 27 when plants are subjected to biotic and abiotic stress, the electric transport chain becomes unbalanced, leading to an increase in the formation of ROS, which are harmful to cells.Following this, the plants immune system protects the cells by using anti-oxidant enzymes including SOD, CAT, and POD 81 .
These findings align with previous research indicating that olive cultivars with elevated levels of antioxidant enzymes have greater resistance to drought 82 and same results for rapeseed 83 brassica napus 84 .Furthermore, the heightened SOD activity seen after the application of ABA indicates the effective functioning of the antioxidant system inducer in protecting plants from oxidative damage.ABA enhances the activities of SOD, glutathione reductase, and catalase, hence reducing the detrimental effects of drought on plants 13,25,27 .
N, P, and K is the most important and abundant nutrient in plants, playing a vital role in growth and development 59 .The application of ABA significantly increased the N, P, and K contents in wheat grains under drought conditions 60 .This enhancement can be attributed to multiple mechanisms: ABA acts as a phytohormone that helps plants respond to drought by reducing water loss and promoting stress tolerance 85 .It also facilitates the uptake and transport of essential nutrients from the soil to the plant, particularly crucial when nutrient availability is limited 86 .Furthermore, Trapeznikov et al. 87 stated that ABA promotes root development and nutrient remobilization, ensuring optimal nutrient levels even under stress.
Lastly, ABA induces the expression of genes related to stress tolerance and nutrient utilization as reported by Wei et al. 88 .In our research, major N-uptake was seen during the grain filling stage, while the lowest was shown under control conditions without drought stress.N absorption during the grain filling stage was greater than the control treatment.The highest values for P-uptake were observed when ABA 2 was applied.The correlation between N uptake and K-uptake is consistent.Correlation analysis revealed that ABA effectively enhances SC, WUE, and chlorophyll content, and mitigates the adverse effects of drought stress.

Conclusion
In the present study, water deficiency had a significant impact on both wheat growth and yield.However, the foliar application of ABA at different wheat growth stages was shown to be effective in improving wheat germination, growth, physiological, water-related characteristics, and yield, even under drought conditions.Specifically, we observed that drought stress during the grain-filling stage resulted in the greatest reduction in grain production.Importantly, we found that ABA application at a concentration of 200 mg/L was the most effective in mitigating the negative effects of drought in wheat.Our findings suggest that the use of ABA could be a valuable tool for enhancing wheat crop yields in dry agricultural systems.Further research is needed to evaluate the effectiveness of such applications under field conditions. https://doi.org/10.1038/s41598-024-71404-4

Fig. 1 .
Fig. 1.Graphical abstract about role of foliar application of Abscisic Acid under drought stress at different growth stages of wheat.

Fig. 2 .
Fig. 2. Illustrates the impact of ABA application on stomatal conductance (m mol −2 s −1 ), water use efficiency (%), and Chlorophyll a and b (mg g −1 FW) contents at critical growth stages of wheat under drought stress.The labels CK, DGFS, DFS, and DTS represent the control as no drought at any stage, drought at grain filling, flowering, and tillering stages, respectively.ABA 0 , ABA 1 and ABA 2 indicates control, 100mgL −1 and 200 mgL −1 ABA application respectively.The error bars in the figure indicate the standard error (n = 3).

Fig. 5 .
Fig. 5. Illustrates the impact of ABA application on Nitrogen (%), Phosphorus (%), and Potassium (%) affected under drought stress at critical growth stages of wheat.The labels CK, DTS, DFS, and DGFS represent the control as no drought at any stage, drought at tillering, flowering, and grain filling stages, respectively.ABA 0 , ABA 1 and ABA 2 indicates control, 100 mgL −1 and 200 mgL −1 ABA application respectively.The error bars in the figure indicate the standard error (n = 3).

Table 1 .
Physicochemical characteristics of the soil.